Because enzymes often bind two substrates cooperatively, it may be difficult to design inhibitors that span both binding sites, using fragment assembly methods. While fragment assembly strategies exploiting a single binding site have yielded many high affinity inhibitors, there are fewer examples of successful fragment assembly across two substrate pockets. We have recently developed and applied NMR methods (1-3) for the design of bi-ligand inhibitors of dehydrogenases, which are a family of two-substrate enzymes. These efforts involved design of one fragment to occupy the NAD(P)H pocket and one to occupy the substrate pocket. While this fragment assembly strategy has yielded high affinity inhibitors (4), it was unusually difficult to engineer high affinity bi-ligands that span both cofactor and substrate sites. This led to the hypothesis that designing inhibitors which span two substrate sites is inherently more difficult, because in addition to binding, ligand fragments must have the interactions needed to drive a conformational change that brings the two sites into proximity and/or properly folds them. These entropic (dynamic) constraints on fragment assembly in two-substrate enzymes need to be better characterized. This application will dissect these effects in an infectious disease drug target (DHPR: dihydrodipicolinate reductase). Project aims are to: 1. Quantify binding synergy between NADH and a substrate analog (PDC, pyridine dicarboxylate) in DHPR, in terms of motion of the NADH nicotinamide ring and of the two protein pockets. Motion (psec-msec) of NADH will be measured using NMR studies of a labeled nicotinamide ring. Binding site residues (backbone 15NH's) will also be analyzed in various complexes, to determine how they change when substrates bind separately or together (is there synergy?). 2. Quantify binding synergy between two fragments of a bi-ligand inhibitor that spans NADH and PDC pockets. Analogous studies to aim 1, but now using fragments of a known bi- ligand inhibitor (Fragment1-linker-PDC). Furthermore, linker (and protein) motion will be compared before and after tethering, to quantify dynamic changes upon tethering. 3. Synthesize bi-ligands between NADH and PDC, then compare effects on affinity and dynamics relative to the Fragment1-PDC bi-ligand. If cofactor is needed to ensure proper formation of the second binding pocket, then NADH should be a more effective Fragment-1. To test this hypothesis, a bi-ligand using NADH in place of our Fragment-1 will be made, then affinity and dynamics of protein and linker compared to results from aims 1 &2. PUBLIC HEALTH RELEVANCE: Enzymes with two binding sites frequently bind their substrates synergistically. These enzymes are often targets of drug design efforts, using fragment assembly. While fragment assembly has had many successes with single-site enzymes, there are fewer successes where fragments were assembled across two substrate pockets. We hypothesize this is because a significant entropic penalty must be overcome to bring together two synergistic sites. This study will quantify this effect in an infectious disease drug target, and determine if fragment assembly is better accomplished using a native cofactor that is able to induce the required conformational changes.